This invention relates to chemical processes of making fractions from North American ginseng (Panax quinquefolium) and compositions containing these fractions. The products of the present invention may be used to stimulate the production of antibodies, or as therapeutics targeted at conditions characterized by low immunity, such as the common cold, influenza, chronic fatigue syndrome, AIDS, cancer, etc. The products of the present invention may also be used as a supplement for cancer patients undergoing chemotherapy or radiation therapy, which is known to cause serious suppression of the immune system.
For hundreds of years, the use of certain non-toxic agents such as herbal compounds has been widely accepted for a variety of physiological conditions, especially in the Orient. Panax ginseng C. A. Meyer is the best known traditional Chinese medicine. The important pharmacological activities of ginseng extracts, alone or in combination with other drugs, include alleviation of renal impairment, inhibition of carcinogenesis and prevention of stress. There are also a number of reports on the influence of ginseng on the immunological responsiveness of the individual. Some immunomodulatory properties that have been reported include enhancement of host resistance against infection, anti-inflammatory effect, inhibition of tumor growth, as well as modulation of some basic immune function at the cellular level.
American ginseng, Panax quinquefolium, is another specie of ginseng which has gained popularity as a health supplement having many beneficial health effects. Several groups of scientists have attempted to isolate and elucidate the structure of the polysaccharides present in ginseng. Some of the polysaccharides have been demonstrated to be active in modulating the immune system.
A series of studies on the isolation, characterization, and biological evaluation of ginseng polysaccharides was carried out by Tomoda""s group in Kyoritsu College of Pharmacy, Japan. In one set of studies, ginseng polysaccharides were fractionated based on their acidity. Two acidic polysaccharides having immunological activities have been isolated from root of Korean ginseng (Panax ginseng)[1,2]. The sliced roots were extracted with hot water. The extract was treated with cetyltrimethylammonium bromide (CTAB) in the presence of sodium H sulfate. The precipitate was separated, dialyzed, and applied to a Sephadex G-25 column, DEAE-Sephacel (Pharmacia) column to give two pure polysaccharides, which were designated as ginsenan PA and ginsenan PB. Gel chromatography on Toyopearl HW-55F gave the values of 1.6xc3x97105 and 5.5xc3x97104 for the molecular weight of ginsenan PA and ginsenan PB, respectively. Quantitative analyses showed that ginsenan PA contained 21.3% arabinose, 53.4% galactose, 2.0% rhamnose, 16.0% galacturonic acid and 2.7% glucuronic acid. The molar ratio of these component sugars was 11:22:1:6:1. Ginsenan PB contained 11.0% arabinose, 32.2% galactose, 8.1% rhamnose, 39.9% galacturonic acid, and 5.0% glucuronic acid. The molar ratio was 3:7:2:8:1. Both polysaccharides showed marked reticuloendothelial system-potentiating activity in a carbon clearance test, and pronounced anti-complementary activity and alkaline phosphatase-inducing activity in a dose dependent manner.
In another study[3], an additional two polysaccharides were isolated from the supernatant of the above extract treated with CTAB, i.e., the supernatant was poured into ethanol. The precipitate was separated and applied to columns of DEAE-Sephadex A-25 and Sephadex G-25 to give another two pure polysaccharides, designated S-IA and S-IIA. Gel chromatography on Toyopearl HW-55F gave the values of 5.6xc3x97104 and 1.0xc3x97105 for the molecular weight of S-IA and S-IIA respectively. Ginsenan S-IA contains 42.3% arabinose, 50.8% galactose and 6.9% galacturonic acid with the molar ratio of 8:8:1. Ginsenan S-IIA is composed of 42.0% L-arabinose, 32.6% galactose, 6.2% glucose, and 19.2% galacturonic acid. The molar ratio is 15:10:2:5.
Several polysaccharide fractions from leaves and roots of Panax ginseng have been separated by Yamada""s group in the Oriental Medicine Research Center of the Kitasato Institute, Japan. The chemical properties and biological activities were investigated and compared.[4]
Ginseng roots and leaves from China, after treated with ethanol to remove their ginsenosides, were extracted with water, and the residue were extracted with 0.5 M NaOH to give water-soluble and alkaline soluble polysaccharide fractions (designated as GR-2 and GL-2 for the water-soluble fractions and GRA-2 and GLA-2 for the alkaline-soluble fractions, respectively). Based on the acidity of the component polysaccharides, all fractions were further fractionated into strongly acidic (designated as GR-3, GL-3, GRA-3, and GLA-3), weakly acidic (designated as GR4, GL4, GRA4, and GLA4), and neutral polysaccharide fractions (designated as GR-5, GL-5, GRA-5, and GLA-5), by treatment with cetyltrimethylammonium. The roots contained larger amount of polysaccharide than the leaves. The strongly acidic polysaccharide fractions from the roots had a high content of uronic acid, even higher than 50%. Similar component sugars were detected from all fractions. They were rhamnose, arabinose, galactose, glucose, galacturonic acid, and glucuronic acid. Galacturonic acid was the main uronic acid component.
The fraction with highest anti-complementary activity, GL-3, was further fractionated with columns of DEAE-Sephadex, Sepharose CL-6B, DEAE Toyopearl, and ethanol precipitation to give fractions designated as GL-PI through GL-PIV.[5] All fractions contained 32-44% uronic acid. Fraction PI had the highest molecular weight of 50,000. PI and PII consisted mainly of Rha, Gal, and GalA, and PIII contained Fuc in addition, whereas PIV consisted of Gal, Glc, and GalA. A detailed structural determination was performed.
An anti-ulcer pectic polysaccharide (GL-BIII) was isolated from weakly acidic polysaccharide fraction GL4 by chromatography on DEAE Sepharose CL-6B and Sepharose CL-6B. It was mainly composed of Rha, Ara, Man, Gal, Glc, GalA, and GlcA in the molar ratio of 3:4:2:10:1:7:4. Detailed structural determination was performed.[6]
Another macrophage Fc receptor expression-enhancing polysaccharide (GL4llb2) was separated from GL4 by anion-exchange chromatography on DEAE-Sepharose CL-6B. Chemical analysis showed that the sample contained 65% total carbohydrate and 33.7% uronic acid. The composition analysis and structural determination were performed.[7]
Another Panax ginseng extract with anticomplementary activity, G-115, was studied by the same group.[8] G-115 was fractionated in order to characterize the active substances for anticomplementary and mitogenic activities. The most potent anticomplementary activity was observed in the crude polysaccharide fraction, G-115G, whereas the water-soluble dialyzable fraction, G-115E, showed the most potent mitogenic activity. G-115G was further purified by precipitation with cetyltrimethylammonium bromide, anion-exchange chromatography on DEAE-Sepharose and gel filtration on Sepharose CL-4B, and a major potent anticomplementary polysaccharide, G-115l1-lla-2-3 was obtained. This polysaccharide was homogeneous. Its molecular weight was estimated to be 3.68xc3x97105. It consisted mainly of arabinose, galactose and glucose in addition to small amounts of galacturonic acid, glucuronic acid and rhamnose.
Ginseng polysaccharides were isolated from Korean, Chinese, and Japanese ginseng by Hikino""s group at the Pharmaceutical Institute, Tohoku University, Japan. The hypoglycemic activities of the ginseng polysaccharides have been tested. The composition and some structure feature have been elucidated.[9--]
Three polysaccharides, quinquefolans A through C, were isolated from American ginseng.[14] Their molecular weights were estimated to be higher than 2.0xc3x97106 by gel chromatography over Sephacryl S-500. The neutral sugar components were mannose and glucose (molar ratio, 1.0:2.3) for quinquefolan A, mannose and glucose (1.0:5.5) for quinquefolan B, and xylose for quinquefolan C. The acidic sugar components in quinquefolans A through C were found to be 10.8, 11.7, and 7.1% respectively. The content of peptide moieties in these glycans was 2.7, 2.9, and 2.3% for quinquefolans A through C respectively. All of these polysaccharides showed hypoglycemic effects in normal and alloxan-reduced hypoglycemic mice.
An acidic polysaccharide with the molecular weight of 150,000, called ginsan, was isolated from Panax ginseng by a research group at the Laboratory of Immunology, Korean Cancer Center Hospital, Seoul, Korea.[15] This polysaccharide was composed of 3.7% protein and 47.1% hexose (glucose and galactose) and 43.1% uronic acid (galacturonic acid). Ginsan induced the proliferation of T cells and B cells and generated lymphokine activated killer cells from both natural killer and T cells through endogenously produced multiple cytokines.[16]
Miao et al. from Northeast Normal University of China has isolated polysaccharides from American ginseng. The purification and structural analysis were performed.[17]
The biological activities of polysaccharides from American ginseng have been investigated by a research group in Norman Bethune University of Medical Science.[18, 19] They found that polysaccharides from American ginseng (PPQ) enhanced lymphocyte transformation. The effect of polysaccharide from Panax quinquefolium (PPQ-1) on cytokine production from murine spleen lymphocyte in vitro was studied. The data suggest that PPQ-1 regulates immune function.
The present inventors have found that certain American ginseng extracts have immunoregulating properties. CVT-E002, and purified fractions PQ2 and PQ223 therefrom, specifically stimulates murine spleen cells to proliferate B cells, which subsequently produce a large amount of antibody. The fractions also increase serum immunoglobulin (e.g., total IgG) levels and stimulate macrophages to produce IL-1, IL-6 and TNF-xcex1. These fractions may be used for the prevention or treatment of general infection and other immune deficiency associated diseases.
Therefore, the present invention is directed to processes of preparing ginseng fractions PQ2, PQ223 and CVT-E002 from samples of American ginseng.
Specifically, a process of preparing ginseng fraction PQ2 comprises:
combining American ginseng with a first solvent comprising an alcohol and heating the resulting solution at a temperature of about 80-100xc2x0 C. for a time period of about 2-4 hours to produce a first ginseng solution;
thereafter separating the first ginseng solution to produce an alcohol/ginseng solution and a first ginseng residue;
thereafter combining the first ginseng residue with water and heating the resulting solution at a temperature of about 80-100xc2x0 C. for a time period of about 2-4 hours to produce a ginseng residue solution;
thereafter separating the ginseng residue solution to produce a second ginseng residue and a first aqueous extract solution containing a first ginseng extract;
providing a second aqueous extract solution which comprises at least a part of the first ginseng extract, wherein in the second aqueous extract solution the proportion of the first ginseng extract to water is about 1:18 to 1:22;
thereafter combining the second aqueous extract solution with a second solvent comprising an alcohol, wherein the proportion of the second solvent to water is about 1:1 to 3:5, to produce a first precipitate and a first supernatant;
thereafter combining the first supematant produced in the previous step with a third solvent comprising an alcohol, wherein the proportion of the third solvent to first supernatant is about 3:2 to 3:1, to produce a second precipitate and a second supernatant; and
isolating the second precipitate to produce ginseng fraction PQ2.
A process of preparing ginseng fraction PQ223 comprises:
providing ginseng fraction PQ2, as described above;
fractionating the ginseng fraction PQ2 to produce a first elution fraction and a second elution fraction, wherein the first elution fraction corresponds to a carbohydrate peak observed between 35 and 50 ml of elution volume and the second elution fraction corresponds to a carbohydrate peak observed between 50 and 85 ml of elution volume, as determined by gel filtration chromatography using the following materials:
(1) a chromatographic column containing a matrix of a spherical cross-linked co-polymer of allyl dextran and N,Nxe2x80x2-methylenebisacrylamide, having a bed dimension of 16xc3x97600 mm, a bed volume of 120 ml, and a fractionation range (MW) of 5000 to 250,000 for globular proteins and 1000 to 80,000 for dextrans, and
(2) an elution buffer of Tris-HCl containing 0.1 N HCl and 0.3 M NaCl at a pH of 7.0; and
isolating and combining the first elution fraction and the second elution fraction to produce ginseng fraction PQ223.
A process of preparing ginseng fraction CVT-E002 comprises:
combining American ginseng with a first solvent comprising an alcohol in a proportion of about 7-9 ml of first solvent per gram of ginseng and heating the resulting solution at a temperature of about 80-100xc2x0 C. for a time period of about 2-4 hours, to produce a first ginseng solution;
thereafter separating the first ginseng solution to produce an alcohol/ginseng solution and a first ginseng residue;
thereafter combining the first ginseng residue with water in a proportion of about 7-9 ml of water per gram of ginseng residue and heating the ginseng residue solution at a temperature of about 80-100xc2x0 C. for a time period of about 2-4 hours, to produce a ginseng residue solution;
thereafter separating the ginseng residue solution to produce a second ginseng residue and an aqueous extract solution containing a ginseng extract; and
drying the aqueous extract solution to produce ginseng fraction CVT-E002.
The present invention also includes ginseng fractions PQ2, PQ223 and CVT-E002, which are prepared according to the processes described above.
The invention further includes ginseng fractions having specific carbohydrate contents.
A first ginseng fraction has a carbohydrate content which comprises about 2-6 mol % rhamnose, about 41-49 mol % galacturonic acid, about 12-18 mol % glucose, about 16-22 mol % galactose and about 12-19 mol % arabinose.
A second ginseng fraction has a carbohydrate content which comprises about 3-8 mol % rhamnose, about 36-44 mol % galacturonic acid, about 2-7 mol % glucose, about 25-33 mol % galactose and about 17-25 mol % arabinose.
A third ginseng fraction has a carbohydrate content which comprises about 0.5-5 mol % rhamnose, about 11-22 mol % galacturonic acid, about 40-60 mol % glucose, about 10-19 mol % galactose and about 11-19 mol % arabinose.
The invention also includes pharmaceutical compositions, comprising the ginseng fractions of the invention, in combination with a pharmaceutically acceptable carrier.
The invention further includes the use of a ginseng fraction according to the invention, alone or in combination with another medicament, in the preparation of a Pharmaceutical composition suitable for treating a condition characterized by low immunity.
The invention also includes the use of a ginseng fraction of the invention to stimulate the production of IL-1, IL-6 and/or TNF-xcex1 in cells.
The invention further includes the use of a ginseng fraction according to the invention to stimulate the in vitro or in vivo production of immunoglobulins.
Also included is the use of a ginseng fraction according to the invention to activate B-lymphocyte proliferation and antibody production therefrom.
The invention also includes a method of treating a condition characterized by low immunity in a patient in need thereof, comprising administering to the patient a condition treating effective amount of a ginseng fraction according to the invention.